Molecular Biology of the Gene Chapter 12

Outline

Genetic Material

Transformation

DNA Structure

Watson and Crick

Eukaryotic DNA Replication

DNA is the Genetic Material

Frederick Griffith investigated virulence of Streptococcus pneumoniae

Two forms of the bacteria:S strain (smooth) has a capsule surrounding the cell, causes mice to die when this strain is injectedR strain (rough) does not have capsule, mice live when this strain injectedHeat-killed S strain, mice liveCombine heat-killed S strain plus live R strain, mice die, live S strain isolated from dead miceConcluded that some substance needed to make the capsule passed from the dead S strain to the living R strain

Virulence passed from the dead strain to the living strainTransformation

Griffith’s Transformation Experiment

Further research by Avery, Macleod, and McCarty

Discovered that DNA is the transforming substance

DNA from dead cell was being incorporated into genome of living cells

Used enzymes:

RNases (breakdown RNA)

Proteases (breakdown proteases)

DNases (breakdown DNA)

Treatment of the transforming substance with DNase prevents transformation

Bacteria and BacteriophagesViruses consist of a protein coat (capsid) surrounding a nucleic acid core.Bacteriophages are viruses that infect bacteria

Hershey and Chase:Radioactively labeled the DNA core and protein capsid of a phage.Results indicated that DNA, not the protein, enters the host.The DNA of the phage contains genetic information for producing new phages.

Hershey and Chase experiments

Determined that DNA is the genetic material

Experiment 1

Viruses with DNA labeled with 32P were incubated with E.coli

Mixed in a blender to remove virus particles attached to cells

Centrifuged so bacteria formed a pellet

Results- viral DNA was inside the bacteria

Hershey and Chase experiments

Experiment 2

Viral proteins in capsids were labeled with 35S and viruses were incubated with E.coli

Mixed in blender and centrifuged

Results- labeled proteins were washed off with the capsids and were not inside the bacteria

Structure of DNA Determined by Watson and Crick

Double helix Composed of monomers called nucleotidesEach nucleotide has 3 parts

Phosphate groupDeoxyribose sugarNitrogenous base- 4 possible

Adenine and guanine- purine bases- double ring structureCytosine and thymine- pyrimidine bases-single ring structure

Erwin Chargaff made key DNA observations that became known as Chargaff’s rule

Rosalind Franklin’s (1920-1958) X-ray diffraction experiments revealed that DNA had the shape of a coiled spring or helix

Purines = Pyrimidines A = T and C = G

X-Ray Diffraction of DNA

In 1953, James Watson and Francis Crick deduced that DNA was a double helix

James Watson (1928- )

Francis Crick (1916-2004)

They came to their conclusion using Tinkertoy models and the research of Chargaff and Franklin

Watson/Crick Model of DNA

Overview of DNA Structure

DNA structure- ladder analogyDNA molecule consists of 2 chains of nucleotides

Arranged in a “ladder” configuration

Alternating phosphate and sugar groups form sides of ladder

Rungs are composed of paired nitrogen bases

Complementary base pairing-purine with pyrimidine

Adenine pairs with thymine

Guanine pairs with cytosine

The “ladder” then coils to form a helix

Eukaryotic Replication

The two DNA strands are held together by weak hydrogen bonds between complementary base pairs

A and TC and G

Each chain is a complementary mirror image of the other

So either can be used as template to reconstruct the other

DNA replication occurs in the nucleus.

Occurs during S phase of interphase of the cell cycle

DNA replication is semi-conservative.

Eukaryotic ReplicationEukaryotic Replication

DNA replication begins at numerous points along linear chromosome.DNA unwinds and unzips into two strands.Each old strand of DNA serves as a template for a new strand.Complementary base-pairing forms new strand on each old strand.

Semiconservative:Each daughter DNA molecule consists of one new chain of nucleotides and one from the parent DNA molecule.The 2 daughter DNA molecules will be identical to the parent molecule.

Enzymes Involved in DNA ReplicationBefore replication begins, the 2 strands of the parent molecule are hydrogen-bonded together.The enzyme DNA helicase unwinds and “unzips” the double-stranded DNA.The enzyme primase puts down a short piece of RNA termed the primer for daughter strand synthesis. RNA primers are removed and replaced with DNA.New DNA nucleotides fit into place along divided strands by complementary base pairing. DNA polymerase- reads along each single strand adding the complementary nucleotideDNA ligase repairs any breaks in the sugar-phosphate backbone.Two daughter DNA molecules have now formed that are identical to the original parent molecule.

Semiconservative Replication of DNA

Old strand –lavenderNew strand- hot pink

Daughter DNA helix Daughter DNA helix

Ladder Configuration and DNA Replication

Meselson and Stahl’sDNA Replication Experiment

Replication Errors

Genetic variations are the raw material for evolutionary change

Mutation:

A permanent (but unplanned) change in base-pair sequence

Some due to errors in DNA replication

Others are due to to DNA damage

DNA repair enzymes are usually available to reverse most errors